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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.29, #3)

The peptide molecular links between the central nervous and the immune systems by I. Z. Siemion; A. Kluczyk; M. Cebrat (pp. 161-176).

The central nervous system (CNS) and the immune system were for many years considered as two autonomous systems. Now, the reciprocal connections between them are generally recognized and very well documented. The links are realized mainly by various immuno- and neuropeptides. In the review the influence of the following immunopeptides on CNS is presented: tuftsin, thymulin, thymopoietin and thymopentin, thymosins, and thymic humoral factor. On the other side, the activity in the immune system of such neuropeptides as substance P, neurotensin, some neurokinins, enkephalins, and endorphins is discussed.

Keywords: Keywords: CNS–immune system molecular links – Immunopeptides – Neuropeptides – Thymic peptides

Almost all about citrulline in mammals by E. Curis; I. Nicolis; C. Moinard; S. Osowska; N. Zerrouk; S. Bénazeth; L. Cynober (pp. 177-205).

Citrulline (Cit, C₆H₁₃N₃O₃), which is a ubiquitous amino acid in mammals, is strongly related to arginine. Citrulline metabolism in mammals is divided into two fields: free citrulline and citrullinated proteins. Free citrulline metabolism involves three key enzymes: NO synthase (NOS) and ornithine carbamoyltransferase (OCT) which produce citrulline, and argininosuccinate synthetase (ASS) that converts it into argininosuccinate. The tissue distribution of these enzymes distinguishes three “orthogonal” metabolic pathways for citrulline. Firstly, in the liver, citrulline is locally synthesized by OCT and metabolized by ASS for urea production. Secondly, in most of the tissues producing NO, citrulline is recycled into arginine via ASS to increase arginine availability for NO production. Thirdly, citrulline is synthesized in the gut from glutamine (with OCT), released into the blood and converted back into arginine in the kidneys (by ASS); in this pathway, circulating citrulline is in fact a masked form of arginine to avoid liver captation. Each of these pathways has related pathologies and, even more interestingly, citrulline could potentially be used to monitor or treat some of these pathologies. Citrulline has long been administered in the treatment of inherited urea cycle disorders, and recent studies suggest that citrulline may be used to control the production of NO. Recently, citrulline was demonstrated as a potentially useful marker of short bowel function in a wide range of pathologies. One of the most promising research directions deals with the administration of citrulline as a more efficient alternative to arginine, especially against underlying splanchnic sequestration of amino acids. Protein citrullination results from post-translational modification of arginine; that occurs mainly in keratinization-related proteins and myelins, and insufficiencies in this citrullination occur in some auto-immune diseases such as rheumatoid arthritis, psoriasis or multiple sclerosis.

Keywords: Keywords: Citrulline metabolism – Urea cycle – Citrullinated proteins – Nitric oxide metabolism – Argininosuccinate synthetase – Ornithine carbamoyltransferase

Teratogenicity of 3-hydroxynorvaline in chicken and mouse embryos by R. Louw; H. C. Potgieter; W. Vorster (pp. 207-212).

3-Hydroxynorvaline (HNV; 2-amino-3-hydroxypentanoic acid), a microbial L-threonine analogue, is toxic to mammalian cells and displays antiviral properties. In view of this, we investigated the toxicity and/or potential teratogenicity of HNV in developing chicken and mouse embryos. HNV was administered to chicken embryos (in ovo; dose 75–300 μmole/egg; 48 h post-incubation) and pregnant Hanover NMRI mice (per os; total dose 900–1800 mg/kg body mass; gestation days 7–9). Control animals received sterile saline solutions. Harvested embryos (chicken embryos, 10 days post-incubation; mouse embryos; gestation day 18) were fixed in glutaraldehyde and stereomicroscopically inspected for signs of dysmorphogenesis. Body mass, body and toe length and mortality of chicken embryos, and the body mass and mortality of mouse embryos were recorded. HNV exposure significantly increased the incidence of embryotoxic (growth retardation, toxic mortality) and congenital defects in both chicken and mouse embryos. All the observed effects were dose-dependent. In conclusion, HNV is an embryotoxic and teratogenic compound, which caused significant developmental delay and congenital defects in developing chicken and mouse embryos.

Keywords: Keywords: 3-Hydroxynorvaline – β-Hydroxynorvaline – Toxic amino acid – Teratogen – Dysmorphogenesis – Chicken embryo – Mouse embryo – Neural tube defects

Apical and basolateral 4F2hc and the amino acid exchange of L-DOPA in renal LLC-PK₁ cells by P. Soares-da-Silva; M. P. Serrão (pp. 213-219).

The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK₁ cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [¹⁴C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [¹⁴C] L-DOPA handling. The basal-to-apical transepithelial flux of [¹⁴C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [¹⁴C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [¹⁴C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [¹⁴C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [¹⁴C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [¹⁴C] L-DOPA through the apical or the basal cell side. It is likely that the Na⁺-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.

Keywords: Keywords: LAT2 – 4F2hc – L-DOPA – LLC-PK₁ cells – Amino acid exchanger

Elimination kinetics of L-alanyl-L-glutamine in ICU patients by A. Berg; O. Rooyackers; Å. Norberg; J. Wernerman (pp. 221-228).

A randomised, double blind, placebo-controlled study was performed giving 0.5 g · kg⁻¹ · day⁻¹ of undiluted alanyl-glutamine (20%) or saline in a peripheral vein during 4 hours in ICU patients (n = 20). During the infusion period a steady state in plasma concentration was reached for alanyl-glutamine, but not for alanine, glutamine or glutamate. On the other hand there was no accumulation of any of the amino acids, as the pre-infusion concentrations were reached within 8 hours after the end of infusion. The half-life of the dipeptide was 0.26 hours (range, 0.15–0.63 h). The distribution volume of alanyl-glutamine was larger than the extracellular water volume, indicating a rapid hydrolysis of the dipeptide. There was no detectable alanyl-glutamine in the urine of any of the patients. All patients had excretion of small amounts of amino acids in urine, but the renal clearance of alanine, glutamine and glutamate were not different between the two groups.

Keywords: Keywords: Vascular tolerance – Glutamine – Parenteral nutrition – Urine – Glutamate – Humans

Expression of LAT1 and LAT2 amino acid transporters in human and rat intestinal epithelial cells by S. Fraga; M. J. Pinho; P. Soares-da-Silva (pp. 229-233).

The present study evaluated the presence of LAT1 and LAT2 amino acid transporters in human Caco-2 cells and rat IEC-6 cells along the mucosa of the rat digestive tract. The LAT1 cDNA was amplified by PCR using two sets of primers (one specific for rat LAT1 and another simultaneously specific for human, rat and mice). The LAT2 cDNA was amplified by PCR using one set of primers simultaneously specific for human, rat and mice LAT2. The presence of LAT1 and LAT2 protein was examined by means of immunoblotting using an antibody raised against the rat LAT1 and mouse LAT2. Caco-2 and IEC-6 cells, as well as the rat intestinal mucosa, are endowed with both LAT1 and LAT2 transporter transcripts and protein. LAT1 protein is most abundant in IEC-6 cells, which is in agreement with functional data previously reported. The findings in the rat intestinal mucosa indicate that LAT1 protein is most abundant in the colon and its abundance markedly decreases at the level of jejunum and ileum, which contrast with relative homogeneous presence of LAT2 across the digestive tract. In conclusion, Caco-2 and IEC-6 cells, as well as the rat intestinal mucosa, are endowed with both LAT1 and LAT2 amino acid transporter transcripts and protein.

Keywords: Keywords: LAT1 – LAT2 – Caco-2 cells – IEC-6 cells – Intestinal epithelium

Analysis of amino acids in individual wheat embryonic protoplast by F.-G. Chen; C. Wang; D.-Y. Zhi; G.-M. Xia (pp. 235-239).

Amino acids analysis in single wheat embryonic protoplast was performed using capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF), combination with tissue culture technique. Reagent fluorescein isothiocyanate (FITC) was introduced into living protoplasts by electroporation for intracellular derivatization. A special osmotic buffer (0.6 mol/L mannitol, 5 mmol/L CaCl₂) was used to keep the osmotic balance of embryonic protoplasts during the protoplasts derivatization. After completion of the derivatization reaction in the protoplasts, a single protoplast was drawn into the capillary tip by electroosmotic flow. Then a 0.1 M NaOH lysing solution was injected by diffusion. The derivatized amino acids were separated by capillary electrophoresis and detected by laser-induced fluorescence detection after the protoplast was lysed Nine amino acids were quantitatively and qualitatively determined and compared in lysate and single protoplast of wheat embryonic cells respectively, with mean concentrations of amino acids ranging from 2.68×10⁻⁵ mol/L to 18.18×10⁻⁵ mol/L in single protoplast.

Keywords: Keywords: Wheat embryonic protoplast – Amino acid – Capillary electrophoresis – Electroporation – Laser-induced fluorescence – Single-protoplast analysis

Synthesis of 1-(N-ethoxycarbonylamino)alkylphosphonic monoesters by H. Liu; J. X. Xu (pp. 241-243).

A series of 1-(N-ethoxycarbonylamino)alkylphosphonic monoesters were synthesized via three-component Mannich-type condensation of ethyl carbamate, aldehydes and dichlorophosphites in benzene, followed by hydrolysis.

Keywords: Keywords: Amino acid – Aminoalkylphosphonic acid – Multiple-component condensation – Mannich-type reaction – Synthesis

Polyamines are absorbed through a y⁺ amino acid carrier in rat intestinal epithelial cells by J. G. Sharpe; E. R. Seidel (pp. 245-253).

Due to the similarity in transport characteristics of polyamines and the y⁺ basic amino acid system, we hypothesized that both substrates could be moving through a common carrier site. Competitive and cross inhibition experiments in intestinal epithelial cells revealed the possibility of a common transport site. N-ethylmalemide (NEM) inhibited both lysine and putrescine transport, confirming that both were carried by a y⁺ transporter. Overexpressing the y⁺ transporter CAT-1 in a polyamine transport-deficient cell line, CHO-MG, did not reconstitute polyamine-transport. Thus, polyamines are not traveling through CAT-1. To determine if lysine is carried by a polyamine transport site, an antizyme-overexpressing cell line was used. Antizyme overexpression decreased polyamine uptake by 50%; in contrast, lysine transport was unaffected. Therefore, lysine is not traveling through a polyamine transport site. It appears that polyamines and lysine are likely traveling through a common unknown y⁺ transport site.

Keywords: Keywords: Basic amino acid – Cationic amino acid – y⁺ transport – Antizyme – Polyamine transport – Putrescine

Potent isozyme-selective inhibition of human glutathione S-transferase A1-1 by a novel glutathione S-conjugate by I. Cacciatore; A. M. Caccuri; A. Cocco; F. De Maria; A. Di Stefano; G. Luisi; F. Pinnen; G. Ricci; P. Sozio; P. Turella (pp. 255-261).

Elevated levels of glutathione S-transferases (GSTs) are among the factors associated with an increased resistance of tumors to a variety of antineoplastic drugs. Hence a major advancement to overcome GST-mediated detoxification of antineoplastic drugs is the development of GST inhibitors. Two such agents have been synthesized and tested on the human Alpha, Mu and Pi GST classes, which are the most representative targets for inhibitor design. The novel fluorescent glutathione S-conjugate L-γ-glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (4) has been found to be a highly potent inhibitor of human GSTA1-1 in vitro (IC₅₀=0.11±0.01 μM). The peptide is also able to inhibit GSTP1-1 and GSTM2-2 isoenzymes efficiently. The backbone-modified analog L-γ-(γ-oxa)glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (6), containing an urethanic junction as isosteric replacement of the γ-glutamyl-cysteine peptide bond, has been developed as γ-glutamyl transpeptidase-resistant mimic of 4 and evaluated in the same inhibition tests. The pseudopeptide 6 was shown to inhibit the GSTA1-1 protein, albeit to a lesser extent than the lead compound, with no effect on the activity of the isoenzymes belonging to the Mu and Pi classes. The comparative loss in biological activity consequent to the isosteric change confirms that the γ-glutamyl moiety plays an important role in modulating the affinity of the ligands addressed to interact with GSH-dependent proteins. The new specific inhibitors may have a potential in counteracting tumor-protective effects depending upon GSTA1-1 activity.

Keywords: Keywords: Glutathione – Glutathione S-transferase inhibitors – Glutathione S-conjugate – γ-Glutamyl transpeptidase – Pseudopeptide

Plasma levels of amino acids in elderly long term care residents with oropharyngeal dysphagia: Comparison of hand-oral with tube-enteral-fed patients by A. Leibovitz; B. Sela; J. Zlotnik; Y. Baumohel; R. Segal (pp. 263-266).

Background: Dysphagia and eating difficulties are highly prevalent in long term care patients. Evaluation of their nutritional status is complicated by comorbidity, frailty and individual patterns of feeding. In previous studies we found vitamin deficiencies (folic acid B6 and B12) in orally fed elderly in early stages of oropharyngeal dysphagia despite satisfactory nutritional parameters (BMI, albumin and hemoglobin). The aim of this study is to evaluate the plasma amino acids levels in these hand-oral fed elderly patients with dysphagia.Methods: Plasma amino acids were measured in 15 orally fed elderly patients in early functional outcome swallowing scale (FOSS), stage 2, and compared with those of 15 matched nasogastric-tube-fed counterparts.Results: The plasma levels of all measured amino acids, ratio of essential to nonessential, levels of conditionally essential and the immune-enhancing amino acids were similar in both groups and within the normal range of our laboratory. The traditional nutritional parameters were also similar in both groups and within the normal range.Conclusions: Plasma levels of amino acids in elderly patients in early stage of FOSS are satisfactory, supporting the view that their protein intake is adequate. Further studies should concentrate on patients in advanced stages of FOSS.

Keywords: Keywords: Plasma amino acids – Oropharyngeal dysphagia – Tube feeding

L-NAME administration prevents the inhibition of nucleotide hydrolysis by rat blood serum subjected to hyperargininemia by D. Delwing; M. C. F. Gonçalves; J. J. F. Sarkis; A. T. S. Wyse (pp. 267-272).

The main objective of the present study was to evaluate the in vivo and in vitro effect of Arg on serum nucleotide hydrolysis. The action of N^ω-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on the effects produced by Arg was also examined. Sixty-day-old rats were treated with a single or a triple (with an interval of 1 h between each injection) intraperitoneal injection of saline (group I), Arg (0.8 g/kg) (group II), L-NAME (2.0 mg/kg or 20 mg/kg) (group III) or Arg (0.8 g/kg) plus L-NAME (2.0 mg/kg or 20 mg/kg) (group IV) and were killed 1 h later. The present results show that a triple Arg administration decreased ATP, ADP and AMP hydrolysis. Simultaneous injection of L-NAME (20 mg/kg) prevented such effects. Arg in vitro did not alter nucleotide hydrolysis. It is suggested that in vivo Arg administration reduces nucleotide hydrolysis in rat serum, probably through nitric oxide or/and peroxynitrite formation.

Keywords: Keywords: Hyperargininemia – Arginine – ATP diphosphohydrolase – 5′ Nucleotidase – Serum

The neuronal differentiation process involves a series of antioxidant proteins by J.-E. Oh; K. Karlmark Raja; J.-H. Shin; M. Hengstschläger; A. Pollak; G. Lubec (pp. 273-282).

Involvement of individual antioxidant proteins (AOXP) and antioxidants in the differentiation process has been already reported. A systematic search strategy for detecting differentially regulated AOXP in neuronal differentiation, however, has not been published so far. The aim of this study was to provide an analytical tool identifying AOXP and to generate a differentiation-related AOXP expressional pattern.The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments: We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical (MALDI-TOF-TOF) identification of proteins to generate a map of AOXP.16 AOXP were unambiguously determined in both cell lines; catalase, thioredoxin domain-containing protein 4 and hypothetical glutaredoxin/glutathione S-transferase C terminus-containing protein were detectable in the undifferentiated cells only. Five AOXP were observed in both, undifferentiated and differentiated cells and thioredoxin, thioredoxin-like protein p19, thioredoxin reductase 1, superoxide dismutases (Mn and Cu-Zn), glutathione synthetase, glutathione S-transferase P1 and Mu1 were detected in differentiated cells exclusively.Herein a differential expressional pattern is presented that reveals so far unpublished antioxidant principles involved in neuronal differentiation by a protein chemical approach, unambiguously identifying AOXP. This finding not only shows concomitant determination of AOXP but also serves as an analytical tool and forms the basis for design of future studies addressing AOXP and differentiation per se.

Keywords: Keywords: Neuronal differentiation – N1E-115 – Antioxidant proteins – Proteomics – Two-dimensional electrophoresis – Matrix assisted laser desorption/ionization-time of flight mass spectrometry

Phenylalanine ammonia-lyase modified with polyethylene glycol: Potential therapeutic agent for phenylketonuria by K. Ikeda; E. Schiltz; T. Fujii; M. Takahashi; K. Mitsui; Y. Kodera; A. Matsushima; Y. Inada; G. E. Schulz; H. Nishimura (pp. 283-287).

Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the defects in the phenylalanine hydroxylase (PAH) gene. Individuals homozygous for defective PAH alleles show elevated levels of systemic phenylalanine and should be under strict dietary control to reduce the risk of neuronal damage associated with high levels of plasma phenylalanine. Researchers predict that plant phenylalanine ammonia-lyase (PAL), which converts phenylalanine to nontoxic t-cinnamic acid, will be an effective therapeutic enzyme for the treatment of PKU. The problems of this potential enzyme therapy have been the low stability in the circulation and the antigenicity of the plant enzyme. Recombinant PAL originated from parsley (Petroselinum crispum) chemically conjugated with activated PEG₂ [2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine] showed greatly enhanced stability in the circulation and was effective in reducing the plasma concentration of phenylalanine in the circulation of mice. PEG-PAL conjugate will be an effective therapeutic enzyme for the treatment of PKU.

Keywords: Keywords: Phenylketonuria – Phenylalanine ammonia-lyase – Phenylalanine – Polyethylene glycol – Enzyme replacement therapy

Alterations in neutrophil (PMN) free intracellular alpha-keto acid profiles and immune functions induced by L-alanyl-L-glutamine, arginine or taurine by J. Mühling; K. A. Nickolaus; M. Halabi; M. Fuchs; M. Krüll; J. Engel; M. Wolff; R. Matejec; T. W. Langefeld; I. D. Welters; T. Menges; M. G. Dehne; A. Sablotzki; G. Hempelmann (pp. 289-300).

The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free α-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN α-ketoglutarate, pyruvate PMN superoxide anion (O₂⁻) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in α-ketoglutarate, pyruvate, MPO release and H₂O₂ generation. Formation of O₂⁻ on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and α-ketobutyrate levels, decreased O₂⁻ and H₂O₂ formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-α-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.

Keywords: Keywords: L-Alanyl-L-glutamine – Arginine – Taurine – Neutrophil – α-Keto acids – Immune function – Superoxide anion – Hydrogen peroxide – Myeloperoxidase

Using string kernel to predict signal peptide cleavage site based on subsite coupling model by M. Wang; J. Yang; K.-C. Chou (pp. 301-301).

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.29, #3)